Process for the production and germination of Entomophthora resting spores

ABSTRACT

Production of Entomophthora resting spores was increased by 50 to 70% by modifying an egg yolk media with a particular maltose agar. Germination of the resting spores at levels up to 100% was obtained by preconditioning harvested spores by treatment with 95% ethanol, high speed blending, sonication, or a combination of ethanol treatment and high speed blending. Germination of spores which had been dried and stored but not pretreated as above was greatly increased by exposure to an atmosphere of 95% ethanol. In addition, treatment by the processes of this invention resulted in the production of a spore stage not previously observed or reported. Spores thus produced have been termed germ conidia.

This is a division of application Ser. No. 681,962, filed Apr. 30, 1976.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the germination of resting spores and moreparticularly to methods for increasing significantly the number orpercentage of resting spores that are germinated.

2. Description of the Prior Art

Entomophthora species are known to cause large scale epizootics andproduce resting spores which might be stored for long periods.Consequently, these fungi are excellent candidates for biologicalcontrol. However, the practical utilization of Entomophthora species asbiological control agents has been hampered by the inability to producelarge quantities of resting spores and induce their germination.Production of resting spores in artificial media and germination of theproduced spores have been the problems.

In the past, many workers such as the following have attempted togerminate or increase the germination of resting spores; Nowakowski,Pamiet. Acad. Umiejejnosci zu Krakau 8, 153-183, 1884; Dustan, Proc.Acad. Entomol. Soc., 9, 14-36, 1924; Gilliatt, Proc. Acad. Entomol.Soc., 10, 46-54, 1925; Schweizer, Planta 35, 132-176, 1947; Hall andHalfhill, Jour. Econ. Entomol., 52, 30-35, 1959; and Krejzova, Jour.Invert. Pathol. 12, 460, 1968 and Ceska Mykol 25, 231-238, 1971.Although Hall and Halfhill reported one experiment in which 15%germination was obtained the level of germination obtained by the aboveworkers was generally less than 5%. French and Gallimore (Agr. Food Chem19, 912-915, 1971) reported on the effect of a number of chemicalcompounds on uredospore germination of wheat stem rust and found some ofthem to have a stimulatory effect. Ethanol, because it was found to beinactive and without any stimulatory effect on germination, was used tosolubilize some of the nonwater-soluble, volatile, oily compounds.French and Weintraub (Arch. Biochem. and Biophys., 72, 235- 237, 1957)has previously reported that extracts of uredospores of wheat stem rustcontained substances capable of inducing germination of the dormantspores. They also found that the extracts contained at least two activecompounds, one of which was pelargaraldehyde (n-nonanal) and showed thatthis compound stimulated germination of wheat rust spores to someextent. However, none of the prior art discloses any means ofgerminating resting spores that approaches the 100% level. Furthermore,none of the known procedures discloses a method or substance that iseasily adaptable to large scale commercial size production andgermination of resting spores.

SUMMARY OF THE INVENTION

It is therefore an object of this invention to provide a process for theproduction and germination of resting spores.

A further object of this invention is to provide a process for theproduction of large quantities of resting spores and for germination ofthese spores at levels up to 100%.

A still further object is to provide a process for producing andgerminating resting spores in quantities adequate for use in thebiological control of insects.

Another object is to provide a process for producing large quantities ofEntomophthora resting spores and for germinating these spores at levelsup to 100% for use in the biological control of aphids.

Still another object is to provide a process for germinating restingspores wherein the resting spores produce spores immediately aftergermination.

According to this invention the above objects are accomplished byproducing the spores on a modified egg yolk media and thenpreconditioning them at harvest time. Standard egg yolk media wasmodified to make the media of this invention by adding molten Sabouraudmaltose agar (SMA) to egg yolk and mixing immediately to prevent lumpingof the agar. Harvested spores were then preconditioned according to thisinvention by contact with ethanol (95%), by high speed blending, bysonication, or by a combination of ethanol contact and high speedblending.

The processes of this invention resulted not only in increasedproduction of resting spores and germination of the spores at levels upto 100%, but also in the production of a spore stage not previouslyobserved. Quite unexpectedly, resting spores of Entomophthorathaxteriana were observed producing spores immediately aftergermination. This spore stage has not been previously observed orreported and, for the purposes of this invention, the spores thusproduced have been termed germ conidia.

DETAILED DESCRIPTION OF THE INVENTION

Cultures of Entomophthora were obtained from diseased aphids collectedfrom potato plants in northern Maine. The most frequently encountedpathogen was E. nr. thaxteriana from the green peach aphid, Myzuspersicae (Sulzer) and the potato aphid, Macrosiphum euphorbiae (Thomas)(Environ. Ethomol 2, 346-347, and 3, 560-562, 1974). Initial isolationwas made on egg yolk media as previously described (Entomophaga 4,261-274, 1959) except that 30 ml. of molten Sabouraud maltose agar (SMA)were added to each 100 ml. of egg yolk. Sabouraud maltose agar is areadily available agar containing the following ingredients per liter:10 gm. polypeptone (a pancreatic digest of casein and a peptic digest ofanimal tissue), 40 gm. maltose, and 15 gm. agar. Modification of thestandard egg yolk media by addition of the molten SMA resulted,unexpectedly, in a great increase in production of resting spores.Insects were collected before eruption of the fungus through thecuticle. These were surface sterilized in streptomycin-penicillin (5,000mcg-5,000 units/ml) and placed directly on the egg yolk media. Theinitial isolates were then transferred to slants of either Sabourauddextrose agar (SDA) or SMA sealed with parafilm and stored at 20° C. toserve as stock cultures for future experiments. Following incubation at25° C. for four to five weeks the medium had been entirely utilized andthe majority of the fungus was in the resting spore state. The contentsof 50 petri dishes were added to 2 liters of water and blended in a 51/2liter blendor. This was diluted again with 2 liters of distilled water,blended with a hand mixer, and centrifuged in a batch rotor in arefrigerated centrifuge once at 23,000 g for 20 minutes, twice at 10,000g for 10 minutes, and once at 2,500 g for 10 minutes. Between each runthe mycelium was easily discernible from the darker colored restingspores. The mycelium, being less dense than the spores, was at thesurface and easily removed. Following initial clean up procedures thespores were re-suspended in distilled water and transferred to 50 mlcentrifuge tubes. A similar procedure was followed as with the batchrotor except that mycelium was removed from the surface of the sporeswith a wash bottle. The purified resting spores were then spread upon150 mm watch glass dishes and air dried. The drying chamber consisted ofa plexiglass box 53 cm × 37 cm × 37 cm. Air was drawn through a descant(silica gel) and passed over the spores. After 18 to 20 hours the sporeswere removed from the dryer, placed in glass containers, and stored at4° C. Standardized resting spore germination tests were used throughoutthe tests. Water agar (2%) was spread evenly over microscope slides andallowed to solidify. These were placed either in desiccators orsupported on glass rods within large petri dishes (150 mm × 15 mm) withdistilled water to maintain 100% relative humidity. Usually one ml ofthe streptomycin-penicillin concentration previously noted was added toeach 100 ml of water agar to prevent bacterial contamination. Normalsterile precautions were used throughout.

The following method was used to determine germination. Resting sporesamples to be tested were placed in suspension in sterile distilledwater (1 mg/ml). The water agar slides were coated with the suspension(approx. 5 ml) and incubated at 25° C. for 48 hours. Germination beganin 12 hours and usually was completed in about 72 hours. However, theproliferation of germ tubes and the disintegration of the emptygerminated spores made determination of germination levels difficultafter 48 hours. By this time, internal changes had taken place withinthe resting spores which were about to germinate. These spores arecharacterized by a densely granular endospore rather than a clear oilglobule. At the end of the test period, the spores were stained withlactophenol aniline blue and a coverslip was added. Random microscopefields were selected until 100 spores had been counted. Eachdetermination was replicated four times. A widefield microscope was usedat 400 × magnification.

The production of spores by the process of this invention just describedwas from 50 to 70% greater than that obtained from egg yolk mediawithout SAM. The spores obtained by this process were then used todevelop procedures for increasing germination.

As previously stated, harvested spores were preconditioned by contactwith ethanol (95%), by high speed blending, by sonication and by acombination of ethanol treatment and high speed blending. In addition,germination was greatly increased by exposing dried spores to anatmosphere of 95% ethanol just prior to being used.

Although many experimental avenues have been tried, the followingprocedure has been adopted for the production and germination of restingspores. The process was developed using Entomophthora spores; however,there is no reason that it should not be applicable to other restingspores.

Media Preparation

1. Surface sterilize hens' eggs in 2.5% sodium-hypoclorite for 30minutes. If the egg white is to be saved, 50% ethanol may besubstituted. It is important to use fresh, unwashed eggs. Eggs that havebeen washed prior to surface sterilization may be contaminated withbacteria.

2. Separate egg yolks aseptically into sterile tall wide-mouthed flasks(500 ml). A small teflon-coated stirring magnet is autoclaved within theflask to facilitate breaking the egg yolks.

3. Add 30 ml of molten SMA to each 100 ml of egg yolk. This must bemixed immediately upon addition to prevent lumping of the agar.

4. Pour media into 10 cm plastic petri dishes at the rate ofapproximately 25 ml per dish.

5. Coagulate the media in an isothermal autoclave at 80° C. for 7minutes. Alternatively, coagulation can be accomplished in an oven at80° C. for 20 minutes. If plastic petri dishes are utilized, it isimportant to note that only certain brands will withstand thistemperature.

If the media is overcooked it becomes leathery and the fungi will notgrow well. This is more likely to occur if dry heat is used forcoagulation. If the media is not coagulated, resting spore formationwill be very poor.

Inoculation of Media

6. Wrap the petri dishes containing the coagulated media in plastic filmto prevent desiccation and store for 3 days to check for contamination.

7. Inoculate the plates by streaking with a sterile needle. The sourceof inoculum should be a vigorously growing culture between 4 and 7 daysold.

8. Wrap the cultures in plastic film, incubate at 25° C. for 7 days, andunwrap. This allows the fungus to cover the entire medium and preventsexcessive desiccation during the incubation period (4 to 5 weeks).

Extraction and Purification

9. Add the contents of 50 petri dishes to 2 liters of water.

10. At this point, one of the following five procedures is followed:

(a) Add ethanol (95%) to make the aqueous culture mixture approximately1% ethanol and mix thoroughly (blend for about 5-10 minutes).

(b) Blend at high speed, approximately 10,000 to 14,000 rpm, for 30minutes.

(c) Add ethanol (95%) to make the aqueous culture mixture approximately1% ethanol and blend at high speed for 30 minutes.

(d) Sonicate for more than one minute.

(e) Mix thoroughly (blend for 5-10 minutes).

11. Store the blended cultures for 24 hours at 4° C. This step is usedonly if step 10(a) has been followed. When steps 10(b), 10(c) and 10(d)or 10(e) are followed, go directly to step 12.

12. Dilute with 2 liters of sterile distilled water, blend with a handmixer, and centrifuge in a batch rotor. Centrifuge under refrigeration,once at 23,000 g for 20 minutes, twice at 10,000 g for 10 minutes, andonce at 2,500 g for 10 minutes. The resting spores are packed tightlyagainst the rotor wall, and the lighter mycelia are easily scraped fromthe surface.

13. Place in 50 ml tubes and centrifuge using angular head at 5,000 rpmfor 10 minutes for final purification. The mycelia are easily washedfrom the surface of the spore pellet with a stream of distilled waterfrom a plastic wash bottle.

14. Spread the pure spores on 150 mm watch glasses and place on rackswithin a plexiglass drying chamber. Air is drawn by fan through silicagel and passed over the spores.

15. Remove the spores after 20 hours and place in sterile containerswith a sachet of silica gel. Store at 4° C. until required.

From the foregoing treatments, the following germination was obtained:step 10(a), 30-40%; step 10(b), 35-55%; step 10(c), 50-100%; step 10(d),30-50%. For step 10(d) a 300 watt sonicator was used.

When spores from step 15, which has been prepared using step 10(e) weremoistened and exposed to an atmosphers of 95% ethanol, 30-40%germination was obtained.

Step 11 was needed when the process followed step 10(a) so that theethanol would be in contact with the culture for a longer period oftime. The 5-10 minute blending in steps 10(a) and 10(e) were needed toput the culture in suspension. However, this was proven not to have anyeffect on germination. Although storage at 4° C. provided the highestrates of germination, it is not a critical temperature. Any temperaturebelow room temperature (20-23° C.) is sufficient.

In view of the fact that French and Gallimore, Supra, found ethanol tobe inactive and not to have any stimulating effect on germination, itwas very surprising and unexpected to obtain 30-40% germination from thepreconditioning treatment with 95% ethanol. The stimulating effect ofsonication was also completely unexpected bacause this procedure isroutinely used to disintegrate cells. However, Entomophthora restingspores not only survived the treatment, but their rate of germinationgreatly increased. It was also found that sonication for at least oneminute provided the desired effect and that sonication for longerperiods of time did not further increase the amount of germination.

Although much of the work developing and verifying the utility of thisinvention was done with Entomophthora thaxteriana, other Entomophthorasuch as E. aphids, E. blullata, E. exitialis, E. grylli, E. virulenta,and an unnamed Entomophthora sp. from spruce budworm Choristoneurafumiferana (Clemens, when treated by the processes of this inventionwere found to yield increases in production of spores and rate ofgermination equal to those of E. thaxteriana and to produce germconidia, the newly discovered spore stage.

I claim:
 1. In a process for germinating resting spores wherein sporesharvested from an inoculated media are put into an aqueous medium andthen extracted, purified, dried and stored, the improvement comprisingpreconditioning the harvested spores prior to extracting and purifyingthem by adding to the spores in the aqueous medium 95% ethanol to makethe aqueous medium approximately 1% ethanol and blending atapproximately 10,000 to 14,000 rpm for 30 minutes.
 2. The improvement ofclaim 1 wherein from 50 to 100% of the spores germinated.
 3. Theimprovement of claim 2 wherein the resting spores produced germ conidiaimmediately after germinating.
 4. In a process for germinating restingspores wherein spores harvested from an inoculated media are put into anaqueous medium and then extracted, purified, dried and stored, theimprovement comprising preconditioning the harvested spores prior toextracting and purifying them by treating them according to a methodselected from the group consisting of (a) adding to the spores in theaqueous medium 95% ethanol to make the aqueous medium approximately 1%ethanol, mixing thoroughly, and storing for about 24 hours; (b) blendingthe spores in the aqueous medium at approximately 10,000 to 14,000 for30 minutes; and (c) sonicating for more than one minute.
 5. Theimprovement of claim 4 wherein from 30 to 55% of the spores germinated.6. The improvement of claim 5 wherein the resting spores produced germcondia immediately after germinating.
 7. In a process for germinatingresting spores wherein harvested spores from an inoculated media are putinto an aqueous medium and then extracted, purified, dried and stored,the improvement comprising moistening the dried spores and then exposingthem to an atmosphere of 95% ethanol prior to germination.
 8. Theimprovement of claim 3 wherein from 30 to 40% of the spores germinated.9. The improvement of claim 8 wherein the resting spores produced germcondia immediately after germinating.